EP3698802A1 - Cellules souches pluripotentes induisant une réparation ostéochondrale - Google Patents

Cellules souches pluripotentes induisant une réparation ostéochondrale Download PDF

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EP3698802A1
EP3698802A1 EP18867476.6A EP18867476A EP3698802A1 EP 3698802 A1 EP3698802 A1 EP 3698802A1 EP 18867476 A EP18867476 A EP 18867476A EP 3698802 A1 EP3698802 A1 EP 3698802A1
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muse
cells
negative
stem cells
pluripotent stem
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EP3698802A4 (fr
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Naosuke KAMEI
Mari Dezawa
Mitsuo Ochi
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Tohoku University NUC
Hiroshima University NUC
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Tohoku University NUC
Hiroshima University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Definitions

  • the present invention relates to a cell preparation and/or pharmaceutical composition used in regenerative medicine. More particularly, the present invention relates to a cell preparation or pharmaceutical composition containing pluripotent stem cells effective for treatment or repair of osteochondral damage.
  • Cartilage lesions cause joint disorders due to limited self-repair ability and a decrease in joint function, and are equivalent to serious disorders particularly in elderly patients (NPL1) [1].
  • stem cells that are expected to have the potential for clinical applications.
  • ES cells embryonic stem cells
  • NSPC neural stem/progenitor cells
  • iPS induced pluripotent stem cells
  • UCBC umbilical cord blood stem cells
  • MSC Mesenchymal stem cells
  • PTL1 adult-derived pluripotent stem cells
  • MSC mesenchymal stem cells
  • Muse cells can be used for improvement and/or treatment of brain damage accompanying fetal growth retardation or that anticipated clinical effects are obtained.
  • An object of the present invention is to provide a novel medical application using pluripotent stem cells (Muse cells) in regenerative medicine. More specifically, an object of the present invention is to provide a cell preparation and/or pharmaceutical composition containing Muse cells for repairing osteochondral damage.
  • Muse cells pluripotent stem cells
  • the inventors of the present invention found that, as a result of injecting or administering Muse cells in an immunodeficient rat model, the Muse cells locally accumulate at sites of osteochondral damage, differentiate into chondrocytes at the damaged site, and are capable of repairing that osteochondral damage, thereby leading to completion of the present invention.
  • the present invention provides as follows.
  • the present invention is capable of repairing osteochondral damage, and particularly subchondral bone covered by fibrous tissue, by an osteochondral tissue regeneration mechanism by which Muse cells differentiate into chondrocytes at a damaged site as a result of directly or intravenously administering Muse cells to the damaged site in a subject suffering from osteochondral damage.
  • the present invention relates to a cell preparation or pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (Muse cells) for treating or repairing osteochondral damage.
  • the present invention can be used to treat, repair or alleviate diseases or symptomatic conditions relating to osteochondral damage such as osteoarthritis, traumatic cartilage damage, rheumatoid arthritis or cancer by using a cell preparation or pharmaceutical composition containing SSEA-3-positive pluripotent stem cells (Muse cells).
  • osteochondral damage refers to damage to bone and/or cartilage attributable to the aforementioned diseases as well as damage occurring as a result of an accident or surgical procedure, although not limited thereto.
  • “Damage” refers to a change in the normal structure or function, has the same meaning as the commonly used term, and can be used synonymously with disorder, degeneration, trauma or defect.
  • bone mainly refers to calcium and phosphoric acid components that have been deposited in the form of hydroxyapatite or collagen (and mainly type I collagen), osteocytes (such as osteoblasts, osteocytes and osteoblasts), and calcified (mineralized) connective tissue including bone marrow formed within true endochondral bone.
  • Bone tissue significantly differs from other tissue (including cartilage tissue). More specifically, bone tissue is vascularized tissue composed from cells and biphasic media (including mineralized and inorganic components (mainly hydroxyapatite crystals) and organic components (mainly type I collagen). Glycosaminoglycans compose less than 2% of these organic components and less than 1% of biphasic media per se or bone tissue per se.
  • the collagen present in bone tissue is present in an extremely organized parallel arrangement in comparison with cartilage tissue.
  • cartilage refers to connective tissue containing chondrocytes, chondrocyte-like cells, intercellular substances (such as type I, type II, type IX and type XI collagen), proteoglycans (such as chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and dermatan sulfate proteoglycans), and other proteins.
  • Cartilage includes articular cartilage and non-articular cartilage.
  • Articular cartilage refers to non-mineralized connective tissue that covers the connecting surface of bone within a joint and fulfills the function as a friction-reducing joint between two opposing bone surfaces. Articular cartilage allows bones move without making direct contact. Articular cartilage partially obtains nutrients from blood vessels of the adjacent periosteum and blood vessels of the bone covered thereby. Articular cartilage contains type II and type IX collagen along with various proteoglycans, but does not contain X type collagen that is related to endochondral osteogenesis. Articular cartilage covers the surface of bone and the bone beneath the cartilage is referred to as subchondral bone.
  • osteochondral damage Damage extending to cartilage and subchondral bone is referred to as osteochondral damage (cartilage full thickness defect), while damage limited to the cartilage layer is referred to as cartilage damage (cartilage partial defect).
  • cartilage damage includes the aforementioned “osteochondral damage” and “cartilage damage”.
  • Non-articular cartilage refers to cartilage not covering a joint surface and includes fibrocartilage (fibrous disc, fibrocartilage disc, connective fibrocartilage and periarticular fibrocartilage) and soft cartilage.
  • fibrocartilage a micropolysaccharide network is combined with protruding collagen bundles and chondrocytes are scattered over a wider range than hyaline cartilage or articular cartilage.
  • the fibrous disc is exposed to impacts and is frequency found in moving joints (such as the meniscus of the knee). Examples of such joints include, but are not limited to, the temporomandibular joint, sternoclavicular joint and knee joint.
  • This fibrocartilage disc which closely adheres to both opposing surfaces, is composed of concentric rings of fibrous tissue having thin layers of cartilage interposed there between.
  • An example this fibrocartilage disc is an intervertebral disc of the spinal cord.
  • Connective fibrocartilage is interposed between the bone surfaces of these joints and is able to move slightly between a vertebral body and between the pubic bone.
  • Periarticular fibrocartilage surrounds the periphery of several articular cavities.
  • the pluripotent stem cells to be used in the cell preparation and pharmaceutical composition of the present invention are typically cells that reside in the human body.
  • the cells which are named "Muse (Multilineage-differentiating Stress Enduring) cells” were discovered by Prof. Dezawa, one of the present inventors.
  • Muse cells can be obtained from bone marrow aspirates and adipose tissue ( Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)) or from skin tissue such as dermal connective tissue, and they are widely dispersed throughout the connective tissue of various organs.
  • the cells have the properties of both pluripotent stem cells and mesenchymal stem cells, and are identified as cells double-positive for the cell surface markers "SSEA-3 (Stage-specific embryonic antigen-3)" and "CD105". Therefore, Muse cells or cell populations containing Muse cells, for example, can be isolated from body tissue using these antigen markers. Muse cells are also stress-tolerant, and can be concentrated from mesenchymal tissue or cultured mesenchymal cells by different types of stress treatments. A cell fraction in which Muse cells were enriched into high content by stress treatment may be used as the cell preparation of the present invention. The methods of separation and identification of Muse cells, and their features, are disclosed in detail in International Patent Publication No. WO2011/007900 .
  • pluripotent stem cells or a cell population containing Muse cells, which were isolated from mesenchymal tissue of a body or cultured mesenchymal tissue by using SSEA-3 as the antigen marker, and which can be used in the cell preparation for treating osteochondral damage (including sequela thereof), may be referred to simply as "SSEA-3 positive cells”.
  • SSEA-3 positive cells pluripotent stem cells
  • non-Muse cells refers to cells that are present in mesenchymal tissue of a body or cultured mesenchymal tissue, and are the remainder of "SSEA-3 positive cells”.
  • Muse cells or a cell population containing Muse cells can be isolated from body tissue (for example, mesenchymal tissue) using only antibody for the cell surface marker SSEA-3, or using antibodies for both SSEA-3 and CD105.
  • body here means "mammalian body”. According to the present invention, the "body” does not include a fertilized ovum or an embryo at a developmental stage before the blastocyst stage, but it does include an embryo at the developmental stage from the blastocyst stage onward, including the fetus or blastocyst.
  • the mammal is not limited to a certain species and may be a primate such as human or monkey, a rodent such as a mouse, rat, rabbit or guinea pig, or a cat, dog, sheep, pig, cow, horse, donkey, goat or ferret.
  • the Muse cells to be used in the cell preparation and pharmaceutical composition of the present invention are clearly distinguished from embryonic stem cells (ES cells) or iPS cells in terms of direct separation from body tissue by using a specified marker.
  • ES cells embryonic stem cells
  • iPS cells in terms of direct separation from body tissue by using a specified marker.
  • the term "mesenchymal tissue” refers to tissue from the bone, synovial membrane, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord or umbilical cord blood, or tissues present in various organs.
  • the Muse cells may be obtained from the bone marrow or skin or adipose tissue.
  • mesenchymal tissue of a body is harvested and the Muse cells are isolated from the tissue and used.
  • the separating means mentioned above may be used to separate Muse cells from cultured mesenchymal cells such as fibroblasts or bone marrow-derived MSCs.
  • the Muse cells to be used for the cell preparation and pharmaceutical composition of the present invention may be either autologous or allogenic with respect to the recipient.
  • Muse cells or a cell population containing Muse cells can be isolated from body tissue by using SSEA-3 positivity, or double positive for SSEA-3 and CD105, as indicators, but human adult skin is known to include various types of stem cells and progenitor cells. However, Muse cells are not identical to these cells.
  • Such stem cells and progenitor cells include skin-derived precursors (SKP), neural crest stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial precursor cells (EP) and adipose-derived stem cells (ADSC). Muse cells can be separated out as being "non-expressing" for the markers unique to these cells.
  • Muse cells can be separated by using non-expression for at least one, and for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 among 11 markers selected from the group consisting of CD34 (EP and ADSC marker), CD117 (c-kit) (MB marker), CD146 (PC and ADSC marker), CD271 (NGFR) (NCSC marker), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snail (SKP marker), Slug (SKP marker), Tyrp1 (MB marker) and Dct (MB marker).
  • CD34 EP and ADSC marker
  • CD117 c-kit
  • MB marker CD146
  • CD271 (NGFR) NCSC marker
  • NG2 PC marker
  • vWF factor von Willebrand factor
  • EP marker Sox10
  • Snail SKP marker
  • Slug SKP marker
  • Tyrp1 MB marker
  • Dct MB marker
  • non-expression of CD117 and CD146 may be used as the indicator for separation
  • non-expression of CD117, CD146, NG2, CD34, vWF and CD271 may be used as the indicator
  • non-expression of all of the aforementioned 11 markers may be used as the indicator for separation.
  • the Muse cells having the aforementioned features to be used for the cell preparation and pharmaceutical composition of the present invention may have at least one property selected from the group consisting of the following:
  • a cell fraction containing Muse cells to be used in the cell preparation of the present invention may be a cell fraction having the SSEA-3 positive and CD105-positive pluripotent stem cells concentrated, obtained by a method of applying external stress treatment to mesenchymal tissue of a body or cultured mesenchymal cells, causing the cells other than the external stress-resistant cells to die, and recovering the surviving cells, the cell fraction having at least one and preferably all of the following properties.
  • the external stress may be any one or a combination of: protease treatment, culturing in a low oxygen concentration, culturing under low-phosphate conditions, culturing with low serum concentration, culturing under low nutritive conditions, culturing under exposure to heat shock, culturing at low temperature, freezing treatment, culturing in the presence of a hazardous substance, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing with agitating treatment, culturing with pressure treatment, or physical impact.
  • the treatment time with a protease is preferably a total of 0.5 to 36 hours to apply external stress to the cells.
  • the protease concentration may be the concentration used when the cells adhering to a culture vessel are detached, when the cell mass is dispersed into individual cells, or when individual cells are recovered from tissue.
  • the protease is preferably a serine protease, aspartic acid protease, cysteine protease, metalloprotease, glutamic acid protease or N-terminal threonine protease.
  • the protease is also preferably trypsin, collagenase or dispase.
  • the cell preparation of the present invention is obtained by suspending Muse cells or a cell population containing Muse cells obtained in the aforementioned (1) in physiological saline or a suitable buffer (such as phosphate-buffered physiological saline).
  • physiological saline or a suitable buffer such as phosphate-buffered physiological saline.
  • cells may be cultured prior to cell transplant and allowed to proliferate until a prescribed cell concentration is obtained.
  • Muse cells do not undergo neoplastic transformation, there is little likelihood of the cells becoming malignant even if cells recovered from biological tissue are contained that have still not differentiated, thereby making them safe.
  • culturing of recovered Muse cells can be carried out in an ordinary growth medium (such as minimum essential medium- ⁇ (a-MEM) containing 10% bovine calf serum). More specifically, a solution containing a prescribed concentration of Muse cells can be prepared by selecting media, additives (such as antibiotics and serum) and the like suitable for the culturing and proliferation of Muse cells with reference to the aforementioned International Publication No. WO 2011/007900 .
  • an ordinary growth medium such as minimum essential medium- ⁇ (a-MEM) containing 10% bovine calf serum.
  • a solution containing a prescribed concentration of Muse cells can be prepared by selecting media, additives (such as antibiotics and serum) and the like suitable for the culturing and proliferation of Muse cells with reference to the aforementioned International Publication No. WO 2011/007900 .
  • DMSO dimethylsulfoxide
  • serum albumin for protecting the cells, or antibiotics and the like for preventing contamination and growth of bacteria
  • other pharmaceutically allowable components such as a carrier, vehicle, disintegrating agent, buffer, emulsifier, suspending agent, soothing agent, stabilizer, storage agent, preservative or physiological saline
  • cells or components other than Muse cells contained in mesenchymal cells may also be contained in the cell preparation.
  • a person with ordinary skill in the art is able to add these factors and pharmaceutical agents to a cell preparation at suitable concentrations. In this manner, Muse cells can be used in the form of a pharmaceutical composition containing various types of additives.
  • the number of Muse cells contained in the cell preparation prepared in the manner described above can be suitably adjusted in consideration of the gender, age and body weight of the subject, disease state and state in which the cells are used so as to obtain the desired effect in treatment of osteochondral damage (such as regeneration of bone and cartilage, disappearance of various diseases related to osteochondral damage, etc.).
  • osteochondral damage such as regeneration of bone and cartilage, disappearance of various diseases related to osteochondral damage, etc.
  • a rat osteochondral defect model with a part of the femur missing, and various types of effects of transplanting Muse cells were examined. Extremely superior effects were obtained by intra-articular administration of SSEA3-positive cells to the rats weighing about 200g to 300g at 5 x 10 4 cells/animal.
  • the cell preparation of the present invention may be administered a plurality of times (such as 2 to 10 times) at a suitable interval (such as twice per day, once per day, twice per week, once per week, once every two weeks, once every one month, one every two months, once every six months) until the desired therapeutic effect is obtained.
  • the therapeutically effective dose is preferably administered, for example, 1 to 10 times at 1 x 10 3 cells to 2 x 10 7 cells per individual.
  • examples of total individual doses include 1 x 10 3 cells to 2 x 10 8 cells, 1 x 10 4 cells to 1 x 10 8 cells, 2 x 10 4 cells to 5 x 10 7 cells, 5 x 10 4 cells to 5 x 10 7 , and 1 x 10 5 cells to 1 x 10 7 cells.
  • the cell preparation of the present invention is not particularly limited to, but may be directly administered to osteochondral damage sites (for example, a joint, a femur, etc.), or may be intravenously administered.
  • a rat osteochondral defect model can be constructed and used in order to study the therapeutic effect of the cell preparation of the present invention on osteochondral damage.
  • rats used as the model typically include, but are not limited to, immunodeficient (such as F344/NJcl-rnu/rnu) Wistar rats and Sprague-Dawley (SD) rats.
  • a rat model was used in which an osteochondral defect was formed in the femur bones of the rats with a metal drill having a spherical tip measuring 1 mm in diameter.
  • the cell preparation of the present invention is in a heterologous relationship with respect to the rats administered the preparation since the preparation consists of human-derived Muse cells.
  • an immunosuppressant such as cyclosporine
  • an immunosuppressant is administered either prior or simultaneous to administration of the heterologous cells in order to suppress a rejection reaction by the heterologous cells within the body.
  • the cell preparation of the present invention is able to restore the function of the bone and cartilage of a patient with osteochondral damage to normal.
  • “restoration” of bone and cartilage function refers to treatment, repair and alleviation of various types of functional disorders accompanying osteochondral damage (including defects), and as an example thereof, refers to alleviation of bone and cartilage damage to a degree that does present a problem during the course of daily life.
  • “Restoration” of bone and cartilage function also refers to a functional disorder caused by osteochondral damage being improved, alleviated or eliminated and returning to the state prior to the osteochondral damage. Histological scoring according to Sellers using safranin O/fast green stain can typically be used to carry out evaluation of restoration of bone and cartilage function as a histological evaluation, although not limited thereto[8].
  • hBMSC Human bone marrow MSC
  • a-MEM Eagle's minimum essential medium
  • FBS fetal bovine serum
  • Glutamax Thermo Fisher Scientific, Waltham, MA
  • the hBMSC were separated into Muse cells (SSEA-3 + ) and non-Muse cells (SSEA-3 - ) corresponding to the presence or absence of expression of SSEA-3.
  • the hBMSC were incubated with SSEA-3 antibody (1:100, Merck Millipore, Darmstadt, Germany) and detected with anti-rat IgM conjugated with allophycocyanin (Jackson Immuno Research, West Grove, PA) in diluted antibody to classify according to FACS Aria II (Becton Dickinson, Franklin Lakes, NJ), which is a special order research project.
  • the present example was carried out with 16 10-week-old immunodeficient rats (32 knees) (F344/NJcl-rnu/rnu). Osteochondral defects (diameter: 2 mm, depth: 2 mm) were formed on the left and right sides within the patellar groove of the femur using a commercially available metal drill having a spherical tip measuring 1 mm in diameter.
  • the rat knees were unevenly distributed among three groups immediately after closing the knee joint (Table 1).
  • the control group was injected with PBS, the non-Muse group was intraarticularly injected with non-Muse cells (5 ⁇ 10 4 cells), and the Muse group was intraarticularly injected with Muse cells (5 ⁇ 10 4 cells). The cells were suspended in 50 ⁇ l of PBS.
  • the rats were sacrificed at 4 weeks and 12 weeks after treatment by intraperitoneal injection of a lethal dose of pentobarbital sodium.
  • the femoral condoyle was evaluated macroscopically using the macroscopic scoring system according to Wayne et al [6][7]. A score of 14 indicates the best result while a score of 0 indicates the poorest result (Table 2).
  • Repaired tissue was fixed for 1 day in phosphate buffer solution (4%) containing paraformaldehyde and decalcified for 4 weeks with 10% EDTA (Nacalai Tesque, Inc., Kyoto, Japan) followed by embedded in a paraffin block.
  • Samples were prepared by cutting 5 ⁇ m sections into a sagittal shape. The sections were stained with safranin O/fast green stain (Muto Pure Chemicals Co., Ltd., Japan) and then subjected to histological scoring according to the Sellers scale for the purpose of histological evaluation (Table 3) [8].
  • Cell density of the repaired tissue was evaluated using hematoxylin and eosin (H & E) stain.
  • Sections were pretreated with antigen recovery reagent (Immunoactive, Matsunami Glass Ind., Osaka, Japan) at 4 weeks and 12 weeks after treatment and immersed in 0.3% H 2 O 2 to block intrinsic peroxidase activity. The sections were then blocked with blocking solution (Protein Block Serum-Free, Dako, Carpinteria, CA) and incubated with mouse monoclonal antibody to type I collagen (1:250, Daiichi Fine Chemical, Toyama, Japan) and type II collagen (1:250, Daiichi Fine Chemical).
  • the visualization reaction was carried out a using avidin-biotin peroxidase system (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA) followed by staining the sections using 3,3'-diaminobenzidine (Peroxidase Substrate Kit, Vector Laboratories, Inc.).
  • Damage borders were easily identified in the patellar grooves of the control and Muse group at 4 weeks and repaired tissue was not detected. Osteoarthritic changes accompanying degeneration of adjacent cartilage increased in the control group in comparison with the Muse group and non-Muse group at 4 weeks and 12 weeks. At 12 weeks, the depth of the damaged portion was filled with brown tissue and decreased in the non-Muse group. In the Muse group, the damaged portion was completely filled with white tissue, this was smooth corresponding to the surrounding tissue, had a uniform surface and it was difficult to clearly identify the damage border ( FIG. 1 ).
  • Table 5 Sellers Scores [Table 5-1] Defect Replenishment relative to Surface of Adjacent Cartilage 4 Weeks 12 Weeks Control Non-Muse Muse Control Non-Muse Muse 2.0 ⁇ 0.7 2.0 ⁇ 0 1.2 ⁇ 0.4* 2.0 ⁇ 0.8 2.2 ⁇ 0.4 1.0 ⁇ 1.1* [Table 5-2] Integration of Repaired Tissue and Peripheral Joint Cartilage 4 Weeks 12 Weeks Control Non-Muse Muse Control Non-Muse Muse 3.0 ⁇ 0 3.0 ⁇ 0 3.0 ⁇ 0 3.0 ⁇ 0 3.0 ⁇ 0 3.0 ⁇ 0 3.0 ⁇ 0 0 0* [Table 5-3] Matrix Staining by Safranin O/Fast Green Stain 4 Weeks 12 Weeks Control Non-Muse Muse Control Non-Muse Muse 4.0 ⁇ 0 4.0 ⁇ 0 4.0 ⁇ 0 4 ⁇ 0 [Table 5-4] Cell Morphology ((a)-(d) selected initially) 4 Weeks 12 Weeks Control Non-Muse Muse Control Non-Muse Muse 5.0 ⁇ 0 5.0+0
  • Intraarticular injection of Muse cells is a promising method for repairing osteochondral damage, and particularly subchondral bone covered by fibrous tissue.

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EP18867476.6A 2017-10-17 2018-10-17 Cellules souches pluripotentes induisant une réparation ostéochondrale Pending EP3698802A4 (fr)

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PCT/JP2018/038687 WO2019078262A1 (fr) 2017-10-17 2018-10-17 Cellules souches pluripotentes induisant une réparation ostéochondrale

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EP3698802A1 true EP3698802A1 (fr) 2020-08-26
EP3698802A4 EP3698802A4 (fr) 2021-05-26

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US9550975B2 (en) 2009-07-15 2017-01-24 Mari Dezawa SSEA-3 pluripotent stem cell isolated from body tissue
US9399758B2 (en) * 2009-07-15 2016-07-26 Mari Dezawa SSEA3(+) pluripotent stem cell that can be isolated from body tissue
JP2014133703A (ja) * 2011-03-30 2014-07-24 Clio Inc 生体組織から単離できるｓｓｅａ−３陽性の多能性幹細胞を含む他家移植用細胞治療用組成物
WO2014027474A1 (fr) * 2012-08-17 2014-02-20 株式会社Ｃｌｉｏ Cellule souche pluripotente induisant une réparation et une régénération après un infarctus du myocarde
WO2015007797A1 (fr) 2013-07-17 2015-01-22 Institut National De La Sante Et De La Recherche Medicale (Inserm) Treillis tridimensionnel fonctionnalisé avec des micro-tissus pour la régénération tissulaire
WO2015064754A1 (fr) * 2013-11-01 2015-05-07 国立大学法人京都大学 Nouveau procédé d'induction de chondrocytes
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US20220313741A1 (en) 2022-10-06
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